Helicobacter pylori is an etiologic agent of a number of gastric disorders in humans, including peptic ulceration and gastric adenocarcinomas. Early efforts to understand H. pylori pathogenesis revealed the presence of a secreted bacterial toxin (VacA) which mounting evidence indicates is an important virulence factor. VacA causes massive degenerative vacuolation of mammalian cells which has been implicated in the gastric epithelial erosion preceding the onset of peptic ulcer disease. The broad objective of this research program si to elucidate the fundamental mechanisms by which VacA mediates cellular cytotoxicity. Long-term milestones include identification of the biochemical activity and intracellular targets of VacA. Experiments in this proposal are designed to test the hypothesis that VacA mediates cellular cytotoxicity as an AB toxin. The model of VacA as an AB toxin is important because it suggests specific hypotheses directly relevant to the fundamental mechanism by which VacA enters host cells and induces vacuolation. The AB family of toxins share a number of distinct properties, and includes prominent members such as cholera, diphtheria, tetanus, and anthrax toxins. The specific aims in this proposal are designed to elucidate the VacA molecular structure and begin to identify structure-function relationships of the toxins. The specific aims are: [1] To identify cellular mechanisms of VacA cytotoxicity. To achieve these goals, novel VacA fusion proteins will be genetically constructed to determine if VacA is trafficked to the cytosol. In addition, radiolabeled VacA will be analyzed for intracellular proteolytic processing. [2] To identify VacA structural characteristics important to the toxin's cellular activities. To achieve these goals, mutant forms of VacA and novel fusion proteins will be genetically constructed to test hypotheses about VacA structure-function relationships and biochemical activities. Because it is estimated that the prevalence of H. pylori infection in developed countries is 20-50%, and 70-90% in developing countries, the importance of developing efficacious vaccines, chemotherapeutics, and diagnostics of H. pylori cannot be overstated. These studies will not only contribute to under understanding of the fundamental mechanisms of VacA-mediated cytotoxicity, but may reveal novel strategies for using attenuated VacA, or fragments of VacA is components in new protective vaccines.